At present there is a great interest in identifying and developing oligonucleotides which are useful in therapy and diagnostics. The use of oligonucleotides in gene therapy aims the inactivation of the genes involved in the process of a disease. There are several strategies of treatments with oligonucleotides.
Antisense therapy uses oligonucleotides with the sequence complementary to the target gene mRNA, which activates a gene silencing mechanism. It can also be used for altering the transcription of the defective gene by modifying, for example, its introns and exons editing pattern.
iRNA small molecules are also used for activating a gene silencing mechanism similar to that of the antisense therapy.
Another possibility is to use oligodeoxyribonucleotides as a decoy for the factors required in the activation of target genes transcription. Transcription factors are bound to the decoys instead of the promoter of the defective gene, which reduces the expression of the target genes. Moreover, single stranded DNA oligonucleotides have been used for directing the shift of one single base inside the sequence of a mutant gene.
On the other hand, nucleic acid fragments with a suitable label (such as DNA probes) are used in diagnostic for the specific hybridization to a nucleic acid to be detected. The specific sequence of the new double strand is visualized with the aid of the label. Thus, genetic, carcinogenic, viral, or diseases caused by other pathogen agents can be detected.
For the applications mentioned above, there are several limitations associated with the targeting to the specific cell, transport across the cell membrane and oligonucleotide stability. In this way, when the oligonucleotide is administered with a therapeutic or diagnostic purpose, sometimes the result obtained is much lower than expected, since it either does not reach the target cell, or it is not able to pass through the membrane, or it breaks down.
In recent years protocols have been developed with the purpose of overcoming such limitations. These protocols are based on conjugating the oligonucleotide to a molecule which targets, specifically, the oligonucleotide to the target cell, which facilitates the transport across the cell membrane or which stabilizes the oligonucleotide. Examples of molecules that can be used with this purpose are, among others, cell penetrating peptides, lipids or polyamines (cf. H. Lönnberg, “Solid-phase synthesis of oligonucleotide conjugates useful for delivery and targeting of potential nucleic acids therapeutics”, Bioconjugate Chem. 2009, vol. 20, pp. 1065-1094; Y. Singh et al., “Chemical strategies for oligonucleotide-conjugates synthesis”, Curr. Org. Chem. 2008, vol. 12, pp. 263-290). Said molecules as well as the labels that can be incorporated into an oligonucleotide are referred to, hereinafter, as “agents”.
Numerous protocols by means of which oligonucleotides are conjugated to agents of the type mentioned above are known in the state of the art. In most of these protocols, one of the steps consists of derivatizing the oligonucleotide with a functional group. This derivatization step is needed for being able to generate, in subsequent steps, the oligonucleotide-agent conjugate. In this derivatization step maleimide may be used as a functional group, thus obtaining maleimide-oligonucleotide derivatives. Maleimide derivatization allows the subsequent conjugation of any agent including a nucleophilic group, such as a thiol, or a diene.
To date, the processes disclosed in the state of the art for preparing maleimide-oligonucleotide derivatives take place in solution (cf. Harrison G. H. et al., “Synthesis and hybridization analysis of a small library of peptide-oligonucleotide conjugates”, Nucleic Acids Res. 1998, vol. 26, pp. 3136-3145; Zanta M. A. et al., “Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus”, Proc. Natl. Acad. Sci. 1999, vol. 96, pp. 91-96). The processes described for obtaining maleimide-oligonucleotide derivatives show regioselectivity problems due to the fact that they are performed in solution. This negatively affects the purity of the resulting maleimide-oligonucleotide derivative and, in turn, the yield of such processes, since the amount of the derivative finally obtained is reduced as being necessary subsequent steps for its purification. The fact that these processes result in derivatives with low yield and purity affects the subsequent steps, wherein the oligonucleotide, functionalized with the maleimide, is conjugated to the agent of interest (peptide, protein, etc.) in such a way that the oligonucleotide has the desired therapeutic or diagnostic effect. Starting from a small amount of the maleimide-oligonucleotide derivative, a much lower final amount of the oligonucleotide with therapeutic or diagnostic activity to that initially expected is obtained.
Therefore, there is a need for providing processes which allow to obtain maleimide-oligonucleotide derivatives with suitable yield and purity.